Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

Tominaga, Keiichiro; Hamada, Yukako

doi:10.1016/j.theriogenology.2003.07.008

Cited by 26 publications

(24 citation statements)

References 34 publications

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“…The mean cost of PCR and electrophoresis using our procedure was close to US$ 2 per sample, which represents a cost reduction of 50-130% using the NaOH extraction method when compared with established DNA extraction kits from renowned suppliers. Using the NaOH-based DNA extraction method (detailed in Figure 2A), an efficient mean sex determination rate for embryos was achieved (95.9%) from a significant number of samples (1847 embryos and embryo biopsies), which is in accordance with other studies on livestock embryo sexing (Macháty et al, 1993;Tominaga and Hamada, 2004) (see Table 2). …”

Section: Discussionsupporting

confidence: 85%

“…In turn, embryo sexing by the PCR-based detection of X-and/or Y-specific regions in retrieved blastomeres is an alternative means of overcoming some of the limitations of the sexed semen for in vivo-or in vitro-produced embryos, allowing the producer to choose which embryo should be transferred to synchronous recipients based on sex determination (Mara et al, 2004;Carneiro et al, 2011;Rattanasuk et al, 2011). The molecular method is efficient for field applications, resulting in pregnancy rates comparable to nonbiopsied embryos, even after cryopreservation (Tominaga, 2004;Tominaga and Hamada, 2004). However, it is frequently the case that the cost of DNA extraction kits, supplies, and reagents in certain regions of the world, or even the impossibility of performing replicates owing to the use of the entire extracted DNA for a single PCR analysis (Lopes et al, 2001;Lopatarova et al, 2010) may limit the wider use of the procedure.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

Tavares¹,

Carneiro²,

Rios³

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

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Section: Discussionsupporting

confidence: 85%

Section: Introductionmentioning

confidence: 99%

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

Tavares¹,

Carneiro²,

Rios³

et al. 2016

Genet. Mol. Res.

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“…In this study, the gender was determined in 91.3% of embryos for fresh transfer and in 87.5% of embryos intended for freezing (Table 1), which accords with our previous work with whole, intact (88.7%; Lopatarova et al, 2007) and splitted embryos (89.4-92%; Lopatarova et al, 2008). Our results are similar to those published by other authors (85-95%) using embryos from superovulated donors (Thibier and Nibart, 1995;Shea, 1999;Li et al, 2007;Yu et al, 2007) as well as IVP embryos (Lopes et al, 2001;Hasler et al, 2002;Tominaga and Hamada, 2004). However, the successful sex detection was inversely related to the number of isolated embryonic cells (≤ 3 cells 85.5%, 4-6 cells 97.4%, ≥ 7 cells 100%; Lacaze et al, 2008).…”

Section: Discussionsupporting

confidence: 91%

Conception rate after sex determination and cryopreservation of D7 bovine embryos:

Lopatářová¹,

Čech²,

Krontorád³

et al. 2010

Vet. Med. - Czech

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ABSTRACT:The aim of this study was to evaluate conception rate after sex determination and subsequent freezing of Day 7 (D 7 ) bovine embryos and to compare it with transfer of fresh female embryos. High quality embryos obtained from superovulated donors were biopsied and the embryonic cells (5-10) were analysed by PCR using specific primers for the Y-chromosome determinant. Fresh embryos were blade biopsied (n = 172) and subsequently transferred (ET) ipsilaterally to synchronized recipients. Selected embryos (n = 112) were biopsied by cell aspiration and cryopreserved for later transfer. Sex determination was successfully completed in 91.3% of fresh group embryos (44% female) and in 87.5% of freeze-thawed group embryos (45.9% female). The achieved pregnancy rate was 56.5% after fresh transfer of sexed (female) embryos whereas intact embryos (control 1) implanted in 61.9% recipients. 95.6% (43/45) of embryos survived biopsy and cryopreservation. After transfer of freeze-thawed sexed (female) embryos, pregnancy was established in 48.8% of recipients. A similar conception rate was found in intact embryos (50.7%, control 2). The results clearly demonstrate that the microsurgical technique used and subsequent PCR sex analysis allow the rapid commercial exchange of genetic resources on the basis of fresh or frozen sex-desired embryos in embryo transfer programs.

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“…However, development of "primer extension preamplification-PCR (PEP-PCR)", which is an in vitro-system for amplifying a large fraction of the DNA sequence present in a single haploid cell [54,55], made it possible to identify the sex of embryos using a single blastomere. The application of PEP-PCR to bovine embryo sexing resulted in a 91% sexidentification rate [56]. The IVP bovine embryos were biopsied on Day-3 or -4 and one or two blastomere samples were subjected to multiplex P C R u si n g p u r i f i e d D N A a f t e r P E P -P C R .…”

Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

Cited by 26 publications

References 34 publications

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

Conception rate after sex determination and cryopreservation of D7 bovine embryos:

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

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